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Means for enabling an immunoassay with a sufficient sensitivity in an
immunoassay for measuring influenza virus in a sample using influenza
virus M1 protein as an antigen is provided. A sample processing method in
an immunoassay for influenza virus, which method comprises, in an
immunoassay for influenza virus using an antibody which undergoes
antigen-antibody reaction with influenza virus matrix 1 protein, or an
antigen-binding fragment thereof, bringing a sample containing influenza
virus into contact with a sample processing liquid containing a
surfactant having at least one group selected from the group consisting
of palmityl, stearyl, and oleyl, is provided.

Inventors:

SHINOHARA; Yuki; (Gosen-shi, JP); MIYAZAWA; Takashi; (Gosen-shi, JP)

Applicant:

Name

City

State

Country

Type

DENKA SEIKEN CO., LTD.

Tokyo

JP

Assignee:

DENKA SEIKEN CO., LTD.TokyoJP

Family ID:

1000001872938

Appl. No.:

14/917632

Filed:

September 10, 2014

PCT Filed:

September 10, 2014

PCT NO:

PCT/JP2014/073971

371 Date:

March 9, 2016

Current U.S. Class:

435/5

Current CPC Class:

G01N 2333/11 20130101; G01N 33/56983 20130101

International Class:

G01N 33/569 20060101 G01N033/569

Foreign Application Data

Date

Code

Application Number

Sep 10, 2013

JP

2013-187355

Claims

1. A sample processing method in an immunoassay for influenza virus, said
method comprising, in an immunoassay for influenza virus using an
antibody which undergoes antigen-antibody reaction with influenza virus
matrix 1 protein, or an antigen-binding fragment thereof, bringing a
sample containing influenza virus into contact with a sample processing
liquid containing a surfactant having at least one group selected from
the group consisting of palmityl, stearyl, and oleyl.

2. The method according to claim 1, wherein said surfactant is a
surfactant having a polyoxyethylene chain.

3. The method according to claim 2, wherein said surfactant is at least
one selected from the group consisting of polyoxyethylene oleyl ether,
polyoxyethylene stearyl ether, and polyoxyethylene cetyl ether.

4. The method according to any one of claims 1 to 3, wherein the final
concentration of said surfactant in said sample processing liquid is
0.005 (w/v) % to 8 (w/v) %.

5. The method according to claim 4, wherein said final concentration is
0.5 (w/v) % to 4 (w/v) %.

6. The method according to claim 1, wherein said sample processing liquid
further contains a chloride.

7. The method according to claim 6, wherein said chloride is an alkali
metal chloride.

8. The method according to claim 6 or 7, wherein the final concentration
of said chloride in said sample processing liquid is 0.05 M to 1.5 M.

9. An immunoassay method for influenza virus, said method comprising
subjecting a sample processed by the method according to claim 1 to an
immunoassay method using an antibody which undergoes antigen-antibody
reaction with influenza virus matrix 1 protein, or an antigen-binding
fragment thereof, to measure influenza virus in said sample.

10. The immunoassay method according to claim 9, which is
immunochromatography.

Description

TECHNICAL FIELD

[0001] The present invention relates to a sample processing method in an
immunoassay for influenza virus using influenza virus matrix 1 protein
(which may hereinafter be referred to as "M1") as an antigen, and an
immunoassay method.

BACKGROUND ART

[0002] Methods such as isolation culture, PCR, ELISA, EIA, Western
blotting, and immunochromatography, are generally known as methods for
detecting influenza virus. Since influenza virus is a seasonal pathogen,
many patients with influenza visit hospitals in a short period, and a
large number of viral tests are carried out in the short period at actual
clinical sites. Therefore, immunochromatography is recently becoming
common at medical sites because of its rapidness and simplicity.

[0003] As common methods of immunochromatography, methods using membranes
such as nitrocellulose membranes are known. In these methods, a ligand
that specifically binds to the substance to be detected is immobilized on
a membrane, and this ligand captures, through the substance to be
detected, a complex containing a labeled substance in which a ligand that
specifically binds to the substance to be detected is labeled, thereby
allowing an assay on the presence or absence of the substance to be
detected in the sample. The labeled substance is generally a ligand which
specifically binds to the substance to be detected and which is labeled
with an enzyme such as alkaline phosphatase, with a colloidal metal such
as colloidal gold, or with a colored polystyrene particle prepared by
staining with a dye. In particular, a colloidal gold particle or a
colored polystyrene particle is used in many cases.

[0004] Most chromatographic methods for detection of influenza virus which
are currently commercially available are based on methods for assaying
the presence or absence of influenza virus in a sample by detection of
nucleoprotein (NP). However, these methods cannot necessarily be said to
have sufficient detection sensitivity, and detection of influenza virus
by these methods is difficult in cases where the sample is derived from a
patient within 6 hours after occurrence of fever. Therefore, further
improvement of the detection sensitivity has been demanded.

[0005] Well-known examples of proteins constituting influenza virus
include HA protein, NA protein, nucleoprotein (NP), and matrix proteins 1
and 2 (M1 and M2). The protein present in the largest number in each
influenza virus particle is M1 protein. The amount of M1 protein is
reported to be about 3 times larger than that of NP protein (Non-patent
Document 1).

[0006] Surfactant treatment of samples to be subjected to immunoassays is
known as described in Patent Document 1 and Patent Document 2.

[0010] The present inventors attempted to construct an
immunochromatography method for detecting influenza virus M1 protein.
However, in immunochromatography for detection of M1 protein wherein a
sample extraction method was carried out using a sample processing liquid
containing a conventionally used surfactant, Triton X100 (trade name) or
Brij35 (trade name), as described in the paragraph 0031 of Patent
Document 1 or the paragraphs 0026 to 0027 of Patent Document 2, the
sensitivity was evidently low, so that the method was impractical (see
Example 4 and Table 5 shown below).

[0011] An object of the present invention is to provide means which
enables an immunoassay with a sufficient sensitivity in an immunoassay
for measuring influenza virus in a sample using influenza virus M1
protein as an antigen

Means for Solving the Problems

[0012] As a result of intensive study, the present inventors discovered
that, by bringing a sample containing influenza virus into contact with a
particular surfactant, the measurement sensitivity of an immunoassay for
influenza M1 protein can be increased, thereby completing the present
invention.

[0013] That is, the present invention provides a sample processing method
in an immunoassay for influenza virus, the method comprising, in an
immunoassay for influenza virus using an antibody which undergoes
antigen-antibody reaction with influenza virus matrix 1 protein, or an
antigen-binding fragment thereof, bringing a sample containing influenza
virus into contact with a sample processing liquid containing a
surfactant having at least one group selected from the group consisting
of palmityl, stearyl, and oleyl. The present invention also provides an
immunoassay method for influenza virus, the method comprising subjecting
a sample processed by the above-described method of the present invention
to an immunoassay method using an antibody which undergoes
antigen-antibody reaction with influenza virus matrix 1 protein, or an
antigen-binding fragment thereof, to measure influenza virus in the
sample.

Effect of the Invention

[0014] By the present invention, in an immunoassay for measuring influenza
virus in a sample using influenza virus M1 protein as an antigen, the
sensitivity of the immunoassay can be increased.

MODE FOR CARRYING OUT THE INVENTION

(Sample)

[0015] The sample to which the present invention is applicable is not
limited as long as the presence of influenza virus in the sample is
suspected. The sample is preferably a nasopharynx-derived sample such as
a nasal swab, nasal aspirate, nasal blow, throat swab, or saliva
(hereinafter referred to as nasopharynx-derived sample). A nasal swab or
a nasal aspirate is especially preferred.

(Sample Processing Liquid)

[0016] As described above, in the method of the present invention, the
sample is brought into contact with a surfactant. This is usually carried
out by treating a sample processing liquid containing the surfactant with
the sample. The sample processing liquid usually contains the surfactant
in a buffer. Examples of the buffer include, but are not limited to, MES,
HEPES, TES, ADA, ACES, bis-Tris, Tris, TES, CAPS, borate buffer,
phosphate buffer, and citrate buffer. The surfactant is a surfactant
having at least one group selected from the group consisting of palmityl,
stearyl, and oleyl. These alkyl groups may be either linear or branched.
The surfactant is preferably a nonionic surfactant in which at least one
of the aliphatic groups is bound to a polyoxyethylene chain. The chain
length of the polyoxyethylene chain is not limited. The degree of
polymerization of oxyethylene units is usually 5 to 40, preferably 10 to
20. Preferred examples of the surfactant having a polyoxyethylene chain
include polyoxyethylene oleyl ether, polyoxyethylene stearyl ether, and
polyoxyethylene cetyl ether. These surfactants may be used individually,
or two or more of the surfactants may be used in combination. The final
concentration of the surfactant in the sample processing liquid (in cases
where a plurality of the surfactants are contained, the total final
concentration of the surfactants) is preferably 0.005 (w/v) % to 8 (w/v)
%, more preferably 0.5 (w/v) % to 4 (w/v) %. The surfactants may be used
individually, or two or more of the surfactants may be used in
combination.

[0017] In the method of the present invention, the surfactant is
preferably brought into contact with the sample in the presence of a
chloride. Therefore, the sample processing liquid preferably further
contains a chloride. Preferred examples of the chloride include alkali
metal chlorides such as lithium chloride, sodium chloride, and potassium
chloride. The final concentration of the chloride in the sample
processing liquid is preferably 0.05 M to 1.5 M, especially preferably
0.1 M to 1.0 M. The chlorides may be used individually, or two or more of
the chlorides may be used in combination.

(Other Components Contained in Sample Processing Liquid)

[0018] The sample processing liquid may also contain one or more of basic
amino acids such as arginine; nonionic surfactants such as
polyoxyethylene octyl phenyl ether (e.g., Triton (registered trademark)
X-100); non-specific reaction inhibitors such as BSA; stabilizers and
preservatives such as sucrose; and antiseptics such as ProClin
(registered trademark). The sample processing liquid may also contain one
or more reagents to be used in the process of adjusting the pH of the
buffer, such as sodium hydroxide or hydrogen chloride.

(Sample Processing Method)

[0019] The sample processing method of the present invention is carried
out by bringing a sample such as a nasopharynx-derived sample into
contact with the sample processing liquid of the present invention. For
example, in cases where the sample is a nasal aspirate, a cotton swab or
the like is soaked in the nasal aspirate, and the cotton swab soaked with
the sample is placed in the sample processing liquid of the present
invention to dissolve the sample into the sample processing liquid. By
this, the sample can be extracted. In cases where the sample is a nasal
swab sample, the nasal cavity is wiped with a cotton swab, and the cotton
swab soaked with the sample is placed in the sample processing liquid of
the present invention to dissolve the sample into the sample processing
liquid. By this, the sample can be extracted. This operation can be
carried out at room temperature. The amount of the sample processing
liquid in which the cotton swab is soaked is not limited as long as the
whole cotton portion in the cotton swab can be soaked in the sample
processing liquid. The amount of the sample processing liquid may usually
be 0.05 mL to 5 mL.

(Immunoassay)

[0020] The sample processed as described above is subjected to an
immunoassay method using an antibody which undergoes antigen-antibody
reaction with influenza virus M1 protein, or an antigen-binding fragment
thereof, to measure influenza virus in the sample. This immunoassay is
described below.

(Virus to be Measured)

[0021] The virus to be measured in the immunoassay of the present
invention is influenza virus such as influenza A virus or influenza B
virus. The "measurement" in the present invention includes any of
detection, quantification, and semi-quantification.

(Antibody)

[0022] The immunoassay of the present invention uses influenza virus M1
protein as an antigen. Therefore, the immunoassay uses an antibody which
undergoes antigen-antibody reaction with influenza virus M1 protein, or
an antigen-binding fragment thereof.

[0023] For the measurement of influenza A virus, an antibody which
undergoes antigen-antibody reaction with influenza A virus M1 (which may
hereinafter be referred to as A-M1), or an antigen-binding fragment
thereof, is used. A-M1 is a protein constituted by 252 amino acid
residues, and its signal due to antigen-antibody reaction can be
specifically detected at a molecular weight of 20 to 35 kD when the
antibody is used for detection by Western blotting. The term "specific"
in the present description means that, in a liquid system containing a
mixture of proteins and the antibody, the antibody does not cause
antigen-antibody reaction with the protein components other than A-M1 at
a detectable level, or, even in cases where the antibody causes a certain
binding reaction or association reaction with a protein component other
than A-M1, the reaction is evidently weaker than the antigen-antibody
reaction between the antibody and A-M1. Amino acid sequences of A-M1 are
known, and described in, for example, GenBank: ACD37490. The anti-A-M1
antibody may be either a monoclonal antibody or a polyclonal antibody.
From the viewpoint of reproducibility, a monoclonal antibody is
preferred. The anti-A-M1 monoclonal antibody can be easily prepared by
immunizing an animal with A-M1 or a partial peptide thereof, and then
carrying out the method of Kohler et al. (Kohler et al., Nature, vol.
256, p. 495-497 (1975)), which is a conventional method. The class of the
monoclonal antibody is not limited to IgG, and may also be IgM or IgY.

[0024] An antigen-binding fragment prepared by isolating only the
antigen-binding site may also be used for the immunoassay. That is,
fragments having specific antigen-binding capacity (antigen-binding
fragments), such as Fab, Fab', F(ab').sub.2, and single-chain antibodies
(scFv) prepared by known methods may also be used.

[0025] For measurement of influenza B virus, an antibody which undergoes
antigen-antibody reaction with influenza B virus M1 (which may
hereinafter be referred to as B-M1), or an antigen-binding fragment
thereof, is used. B-M1 is a protein constituted by 248 amino acid
residues, and its signal due to antigen-antibody reaction can be
specifically detected at a molecular weight of 20 to 35 kD when the
antibody is used for detection by Western blotting. Amino acid sequences
of B-M1 are known, and described in, for example, GenBank: AEN79424.
Similarly to the cases of type A, an antigen-binding fragment may be used
also for influenza B virus.

[0026] The immunoassay per se is well known, and any of sandwich methods,
agglutination methods, competition methods, and various well-known
immunoassays may be employed. Among these, sandwich methods are
preferred. Among sandwich methods, immunochromatography or ELISA is
preferred. Immunochromatography is especially preferred from the
viewpoint of simplicity of the operation. The sandwich method is carried
out using two kinds of monoclonal antibodies capable of binding to the
antigen, influenza virus M1 protein, at the same time, or antigen-binding
fragments thereof. The immunochromatography, which is a preferred
sandwich method, is described below. Immunochromatography per se is well
known and widely used. A well-known method for immunochromatography may
be used also in the present invention.

(Immunochromatography for Detection of Substance to be Detected)

[0027] The method of immunochromatographic detection of the substance to
be detected in the present invention (immunochromatography) is not
limited as long as it is an immunological detection method using an
antibody against the substance which is to be detected (which may
hereinafter be referred to as an antibody against the substance to be
detected). The method is preferably a sandwich method using an antibody
against the substance to be detected and a labeled antibody against the
substance to be detected. The antibody against the substance to be
detected may be either a polyclonal antibody or a monoclonal antibody. A
monoclonal antibody is preferred. The immunochromatography is carried out
by bringing a substance to be detected, extracted by the sample
processing method described above, into contact with a strip or the like
on which the antibody against the substance to be detected is
immobilized. Needless to say, the immunochromatography in the present
invention can be used for both qualitative detection and quantitative
measurement.

[0029] The colored latex particles can be prepared by, for example,
preparing polystyrene particles by soap-free polymerization without using
an emulsifier according to the method described in [0022] of JP 6-306108
A, and then carrying out the method described in [0025] to [0035] of the
same document. Alternatively, colored particles which are commercially
available from, for example, Seradyn Inc. or Magsphere Inc. may be used.

[0030] Cases where colored latex particles are used as the labeling
substance are described below in detail.

(Method for Immobilization of Labeled Antibody)

[0031] The immobilization of the antibody on the colored latex is usually
carried out by chemical bonding. The antibody concentration in this
process is preferably 1 mg/mL to 5 mg/mL, and the buffer and the pH are
preferably 20 mM MES buffer (pH 5.5 to 6.5) or 50 mM borate buffer (pH 8
to 9), more preferably 20 mM MES buffer (pH 6.5). The areas on the
colored latex particles where the antibody is not bound are preferably
blocked by binding of BSA or the like. The thus prepared colored
latex-labeled antibody is stored as a dispersion in a preservation
reagent which inhibits denaturation. Examples of the denaturation
inhibitor include proteins such as BSA; glycerin; and sugars.

(Solid Phase)

[0032] Examples of the material of the solid phase include polyethylene;
polyethylene terephthalate; nylons; glasses; polysaccharides such as
cellulose and cellulose derivatives; and ceramics. Preferred specific
examples of the material of the solid phase include glass fiber filter
papers, cellulose filter papers, and the like commercially available
from, for example, Millipore, Toyo Roshi Kaisha, Ltd., Whatman, and
Rydell; polystyrene plates; glass fiber membranes; nylon membranes; and
nitrocellulose membranes. Nitrocellulose membranes are especially
preferred. Cases where a nitrocellulose membrane is used as the material
of the solid phase are described below.

(Immobilization of Capture Antibody on Solid Phase)

[0033] The capture antibody for detection of the complex of the antigen as
the substance to be detected (for example, influenza virus) and the
labeled antibody may be immobilized on a nitrocellulose membrane by a
generally well-known method. For example, in cases of a lateral-flow
type, an apparatus having a mechanism by which a liquid containing the
capture antibody can be discharged from a nozzle at a constant rate while
the apparatus horizontally moves is used to linearly apply the capture
antibody liquid to a cellulose membrane. The concentration of the
antibody in this process is preferably 0.1 mg/mL to 5 mg/mL, more
preferably 0.5 mg/mL to 2 mg/mL. Normally, the antibody liquid can be
prepared using a predetermined buffer. Examples of the type of the buffer
include normally used buffers such as phosphate buffer, Tris buffer, and
Good's buffer. The buffer preferably has a pH within the range of 6.0 to
9.5, more preferably 6.5 to 8.5, still more preferably 7.0 to 8.0. The
buffer may also contain one or more of salts such as NaCl; stabilizers
and preservatives such as sucrose; and antiseptics such as ProClin
(registered trademark). Examples of the salts include not only those to
be included for adjustment of the ionic strength, such as NaCl, but also
those to be present during the process of adjusting the pH of the buffer,
such as sodium hydroxide.

[0034] After the immobilization of the antibody on the nitrocellulose
membrane, the membrane may be coated with a normally used blocking agent
in the form of a solution or a vapor, to carry out blocking.

[0035] By appropriately selecting the pore size of the nitrocellulose
membrane, the flow rate of the immune complex of the
colored-latex-labeled antibody and the antigen as the substance to be
detected (for example, influenza virus) in the membrane can be
controlled. Since, by controlling this flow rate, the amount of the
labeled antibody bound to the antibody immobilized on the membrane can be
controlled, it is preferred to select a membrane having an appropriate
pore size. Hi Flow Plus HF180, manufactured by Millipore, or the like is
preferably used.

(Immunochromatography Reagent, and Immunochromatography Reagent Kit)

[0036] The sample processing liquid of the present invention may be used
together with a conventional immunochromatography reagent(s), or these
may be used in combination as an immunochromatography reagent or an
immunochromatography reagent kit.

[0037] The "immunochromatography reagent" includes one or more of reagent
components necessary for the measurement by immunochromatography, and
members such as test strips.

[0038] Essentially, in the present invention, it can be said that the
presence of the particular surfactant mentioned above, or the presence of
the particular salt in addition to the particular surfactant, in the
immunochromatographic system for detection of influenza virus is
important for highly sensitive detection. In view of this, Examples are
described below for cases where the method for processing influenza virus
is carried out with a sample processing liquid prepared in a liquid
state. However, the present invention is not limited to the Examples, and
any mode in which an immunochromatographic system for detection of
influenza virus contains the particular surfactant mentioned above, or
the particular salt in addition to the particular surfactant, is within
the scope of the present invention.

EXAMPLES

1. Preparation of Anti-Influenza B Virus M1 Monoclonal Antibodies

[0039] BALB/c mice were immunized with an influenza B virus antigen, and
kept for a certain period. From each mouse, the spleen was removed, and
fusion with mouse myeloma cells (P3.times.63) was carried out by the
method of Kohler et al. (Kohler et al., Nature, vol. 256, p. 495-497
(1975)). The resulting fused cells (hybridomas) were kept in an incubator
at 37.degree. C. The cells were then purified (into monoclonal cells)
while the antibody activity in the supernatant was checked by ELISA using
a plate on which an influenza B virus M1 antigen is immobilized. Each of
two cell lines obtained was intraperitoneally administered to
pristane-treated BALB/c mice. About two weeks later, antibody-containing
ascites was collected. From the ascites obtained, IgG was purified by
affinity chromatography using a protein A column. By this, two kinds of
purified anti-influenza B virus M1 antibodies were obtained.

[0040] A liquid prepared by diluting the purified anti-influenza B virus
M1 antibody with purified water to 1.0 mg/mL was linearly applied to a
predetermined position of a nitrocellulose membrane lined with a PET
film. The membrane was then dried at 45.degree. C. for 30 minutes to
obtain a membrane on which the anti-influenza B virus M1 antibody is
immobilized (hereinafter referred to as "antibody-immobilized membrane".

[0041] The other purified anti-influenza B virus M1 antibody, which was
not used for the immobilization on the nitrocellulose membrane, was
diluted with purified water to 1.0 mg/mL, and colored polystyrene
particles were added to the resulting dilution at 0.1%. The resulting
mixture was stirred, and carbodiimide was then added thereto at 1%,
followed by further stirring the mixture. The supernatant was removed by
centrifugation, and the precipitate was resuspended in 50 mM Tris (pH
9.0) supplemented with 3% BSA, to obtain colored polystyrene particles to
which the anti-influenza B virus M1 antibody is bound.

[0042] A predetermined amount, 1.0 .mu.g, of the colored polystyrene
particles to which the anti-influenza B virus M1 antibody is bound
obtained in 3 were applied to a glass-fiber non-woven fabric, and the
non-woven fabric was then dried at 45.degree. C. for 30 minutes.

5. Lamination with Antibody-Immobilized Membrane, Dry Pad, and Other
Members

[0043] The antibody-immobilized membrane and the dry pad prepared in 2 and
4 were laminated with other members (backing sheet, absorption zone, and
sample pad), and the resulting laminate was cut into a piece with a width
of 5 mm, to provide an influenza B virus test piece.

6. Preparation of Anti-Influenza A Virus M1 Monoclonal Antibody

[0044] BALB/c mice were immunized with an influenza A virus antigen, and
kept for a certain period. From each mouse, the spleen was removed, and
fusion with mouse myeloma cells (P3.times.63) was carried out by the
method of Kohler et al. (Kohler et al., Nature, vol. 256, p. 495-497
(1975)). The resulting fused cells (hybridomas) were kept in an incubator
at 37.degree. C. The cells were then purified (into monoclonal cells)
while the antibody activity in the supernatant was checked by ELISA using
a plate on which an influenza A virus M1 antigen is immobilized. Each of
two cell lines obtained was intraperitoneally administered to
pristane-treated BALB/c mice. About two weeks later, antibody-containing
ascites was collected. From the ascites obtained, IgG was purified by
affinity chromatography using a protein A column. By this, two kinds of
purified anti-influenza A virus M1 antibodies were obtained.

7. Immobilization of Anti-Influenza A Virus Antibody on Nitrocellulose
Membrane

[0045] A liquid prepared by diluting the purified anti-influenza A virus
M1 antibody with purified water to 1.0 mg/mL was linearly applied to a
predetermined position of a nitrocellulose membrane lined with a PET
film. The membrane was then dried at 45.degree. C. for 30 minutes to
obtain a membrane on which the anti-influenza A virus M1 antibody is
immobilized (hereinafter referred to as "antibody-immobilized membrane".

[0046] The other purified anti-influenza B virus M1 antibody, which was
not used for the immobilization on the nitrocellulose membrane, was
diluted with purified water to 1.0 mg/mL, and colored polystyrene
particles were added to the resulting dilution at 0.1%. The resulting
mixture was stirred, and carbodiimide was then added thereto at 1%,
followed by further stirring the mixture. The supernatant was removed by
centrifugation, and the precipitate was resuspended in 50 mM Tris (pH
9.0) supplemented with 3% BSA, to obtain colored polystyrene particles to
which the anti-influenza A virus M1 antibody is bound.

9. Application/Drying of Colored Polystyrene Particles to which
Anti-Influenza A Virus M1 Antibody is Bound

[0047] A predetermined amount, 1.0 .mu.g, of the colored polystyrene
particles to which the anti-influenza A virus M1 antibody is bound
obtained in 8 were applied to a glass-fiber non-woven fabric, and the
non-woven fabric was then dried at 45.degree. C. for 30 minutes.

10. Lamination with Antibody-Immobilized Membrane, Dry Pad, and Other
Members

[0048] The antibody-immobilized membrane and the dry pad prepared in 7 and
9 were laminated with other members, backing sheet, absorption zone, and
sample pad, and the resulting laminate was cut into a piece with a width
of 5 mm, to provide an influenza A virus test piece.

Example 1

Selection of Optimal Type of Surfactant

[0049] In order to search for an optimal surfactant for
immunochromatography for detecting influenza A and B virus M1 proteins,
performances of surfactants were compared. First, sample processing
liquids each containing one of the surfactants shown in Table 1 at a
concentration of 1 (w/v) %, and also containing 10 mM MES (pH 7.0) and 3%
BSA as other components, were prepared. The sample processing liquid
containing No. 4, polyoxyethylene octyl phenyl ether, in Table 1,
corresponds to a conventional sample processing method. Table 1 also
shows the carbon number of the polyoxyethylene (POE) chain of each
surfactant used. For the tests for influenza A virus, Nos. 1 to 7 in
Table 1 were used. For the tests for influenza B virus, only Nos. 2 to 4
in Table 1 were used.

[0050] Subsequently, 30 .mu.L of inactivated influenza A virus or
inactivated influenza B virus was added to 400 .mu.L of each of the
sample processing liquids prepared, and each resulting mixture was mixed.
To the sample pad portion of the influenza A or B virus test piece, 50
.mu.L of the mixture was added dropwise, and visual judgment was carried
out 10 minutes later. Cases where a signal could be found on the test
line were evaluated as "+". Each signal was evaluated as "2+", "3+",
"4+", or "5+", in that order, as the signal intensity increased (Table 2
shows the results).

[0051] As a result, it was found that high signal intensity can be
observed in sample processing methods using a surfactant whose carbon
number in the linear chain of the alkyl group is not less than 16.

[0052] The influence of the concentration of polyoxyethylene cetyl ether
on the immunochromatography for detection of influenza A or B virus M1
protein was investigated. Sample processing liquids each containing
polyoxyethylene cetyl ether at a concentration shown in Table 3-1, and
also containing 10 mM MES (pH 6.5) and 3% BSA as other components, were
prepared. As a control, a sample which does not contain polyoxyethylene
cetyl ether was provided (No. 1 in Table 3-1 and Table 3-2).

[0053] Subsequently, 30 .mu.L of inactivated influenza A virus or
inactivated influenza B virus was added to 400 .mu.L of each of the
sample processing liquids prepared, and each resulting mixture was mixed.
To the sample pad portion of the influenza A or B virus test piece, 50
.mu.L of the mixture was added dropwise, and visual judgment was carried
out 10 minutes later. Cases where a signal could be found on the test
line were evaluated as "+". The signal was evaluated as "2+", "3+", "4+",
or "5+", in that order, as the signal intensity increased (the results
are described in Table 2). ".+-." indicates that a weak signal was found.

[0054] As a result, for both type A and type B, signals stronger than that
for the control condition (No. 1) could be found at polyoxyethylene cetyl
ether concentrations within the range of 0.005 to 8 (w/v) %.

[0055] The effects of addition of salt components on the
immunochromatography for detection of influenza A or B virus M1 protein
were investigated. First, sample processing liquids each containing 1
(w/v) % polyoxyethylene cetyl ether, one of sodium chloride, potassium
chloride, and lithium chloride as a salt component at a concentration
shown in Table 7, and 10 mM MES (pH 7.0) and 3% BSA as other components,
were prepared. For control conditions, sample processing liquids which
are the same as those described above except that salts are not contained
were also prepared. Subsequently, 30 .mu.L of inactivated influenza A
virus or inactivated influenza B virus was added to 400 .mu.L of each of
the sample processing liquids prepared, and each resulting mixture was
mixed. To the sample pad portion of the influenza B virus test piece, 50
.mu.L of the mixture was added dropwise, and visual judgment was carried
out 10 minutes later. Cases where a signal could be found on the test
line were evaluated as "+". The signal intensity is expressed using a
relative value such as "2+", "3+", "4+", or "5+", wherein the value
increases as the signal intensity increases (Table 4-1 to Table 4-3 show
results on type A, and Table 5-1 to Table 5-3 show results on type B).

[0056] As a result, slight increases in the signal intensity could be
observed in the cases where a sample processing liquid supplemented with
a chloride was used. In the cases of potassium chloride or lithium
chloride, further slight increases in the signal intensity could be
observed relative to other chlorides. It was found that a further
increase in the signal intensity can be achieved with a sample processing
liquid containing a mixture of potassium chloride and lithium chloride
(No. 7 in Table 7) compared to cases where these are used individually.
Although data are not shown, use of sodium thiocyanate caused
non-specific reaction in some cases. It was therefore suggested that the
type of the salt is important.

[0057] From the above results, it was discovered that the signal intensity
in the immunochromatography for detection of M1 can be increased by
adding a salt such as sodium chloride, potassium chloride, or lithium
chloride, and using the salt at a concentration of 0.05 M to 1.5 M.

Study on Sensitivity-Increasing Effect of Sample Processing Method for M1
Detection Using Optimal Combination of Surfactant and Salt in
Immunochromatography for Detection of Influenza A or B Virus M1

[0060] As the sample processing liquid for detection of M1, a sample
processing liquid containing 2% polyoxyethylene cetyl ether and 1%
polyoxyethylene octyl phenyl ether, and also containing 0.25 M potassium
chloride and 0.25 M lithium chloride as salt components, and 10 mM MES
(pH 7.0) and 3% BSA as other components, was prepared. To provide a
control condition, a sample processing method containing 10 mM MES (pH
7.0), 3% BSA, and 1% polyoxyethylene octyl phenyl ether was prepared. The
two kinds of sample processing methods described above were subjected to
performance tests by immunochromatography using a dilution series
(undiluted liquid, and up to 1024-fold dilutions) of influenza A or B
virus. At the same time, comparison with conventional
immunochromatography for detection of NP was carried out.

[0061] As a result, in the detection of influenza A virus, detection of
the diluted sample prepared by 1024-fold dilution of the undiluted
inactivated influenza A virus liquid was possible with the sample
processing liquid for detection of M1, while detection of only the
undiluted inactivated influenza A virus liquid was possible with the
control sample processing liquid (Table 6-1). In the Table, the following
symbols are used for representing the judgment results: "+", a signal
could be found on the test line; ".+-.", the reaction could be found
although the signal was weak; "-", no signal could be found.

[0062] In the detection of influenza B virus, detection of the diluted
sample prepared by 512-fold dilution of the undiluted inactivated
influenza B virus liquid was possible with the sample processing liquid
for detection of M1, while detection of only the undiluted inactivated
influenza B virus liquid was possible with the control sample processing
liquid, similarly to the cases of type A (Table 6-2).

[0063] In the immunochromatography for detection of M1 protein, not less
than two times higher sensitivity could be observed for each of influenza
A and B viruses compared to conventional immunochromatography for
detection of NP (Table 6-1, Table 6-2).

[0064] From the above results, it was discovered that, as a sample
processing method in immunochromatography for detection of influenza
virus M1 protein, a sample processing method containing, as a surfactant
component, a surfactant having a palmityl group, stearyl group, or oleyl
group, and containing, as a salt component, potassium chloride, lithium
chloride, or sodium chloride, is effective.

Table 6. Study on Sensitivity-Increasing Effect of Sample Processing
Method for M1 Detection Using Optimal Combination of Surfactant and Salt
in Immunochromatography for Detection of Influenza A or B Virus M1